Background The aim of this study was to improve fluorescence laparoscopy of pancreatic cancer in an orthotopic mouse model with the use of an LED light source and an optimal fluorophore combination. conjugated with Alexa 488 or Alexa 555. Cancer lesions were detected and localized under each light mode. Intravital images were obtained with the OV-100 Olympus Small Animal Imaging System and Maestro CRI Small Animal Imaging System serving as a positive control. Tumors were collected for histologic review. Results Fluorescence laparoscopy with a 495-nm emission filter and an LED light source enabled real-time visualization of the fluorescence-labeled tumor deposits in the peritoneal cavity. The simultaneous use of different fluorophores (Alexa 488 and Alexa 555) conjugated to antibodies brightened the fluorescence signal enhancing detection of sub-millimeter lesions without compromising background illumination. Adjustments to the LED light source permitted simultaneous detection of tumor lesions of different fluorescent colors and surrounding structures with minimal autofluorescence. Conclusions Using an LED light source with adjustments to the red blue and green wavelengths we can simultaneously identify tumor metastases expressing fluorescent proteins of different wavelengths which greatly enhanced the signal without compromising background illumination. Development of this technology for clinical use can improve staging and treatment of pancreatic cancer. nude mice were maintained in a barrier facility on high-efficiency particulate air filtered racks and fed with autoclaved laboratory rodent diet (Teckland LM-485; Western ResearchProducts Orange CA). All surgical procedures were performed under anesthesia with an intramuscular 20(R)Ginsenoside Rg2 injection of 100 μL of a mixture of 100 mg/kg ketamine and 10 mg/kg xylazine. All animal studies were approved by the UCSD Institutional Animal Care and Use Committee (IACUC) and conducted in accordance with the principles and procedures outlined in the National Institutes of Health (NIH) Guide for the Care and Use of Animals. Orthotopic model Human pancreatic cancer cells (106) were implanted orthotopically in the tail of the pancreas as previously described.18 The following orthotopic models were established: SNX14 BxPC-3 20(R)Ginsenoside Rg2 labeled with Alexa 488-conjugated anti-CEA antibody or labeled with Alexa 555-conjugated anti-CEA antibody; FG-RFP; FG-RFP co-implanted with BxPC-3 labeled with Alexa 488-conjugated anti-CEA antibody BxPC-3-RFP labeled with Alexa 20(R)Ginsenoside Rg2 488-conjugated anti-CEA antibody MiaPaca2-GFP co-implanted with BxPC-3 labeled with Alexa 555-conjugated anti-CEA antibody. The orthotopic models used in the present study involved injection of pancreatic cancer cells into the tail of the pancreas is an exact orthotopic model allowing spontaneous metastases. A splenic injection would allow metastasis to the liver but is not exactly orthotopic and allows for what are termed “experimental metastasis” to the liver.24 Carcinomatosis model Human pancreatic cancer cells were resuspended at 20(R)Ginsenoside Rg2 a concentration of 1×106 cells per 100 μL of serum- free medium and placed on ice before intra-peritoneal injections directly into the peritoneal cavity of 6-week-old female nude mice using a 27-gauge needle as previously described.19 Fluorescence Laparoscopy A standard laparoscopic tower provided by Stryker (Stryker San Jose California) was slightly modified in the following manner to achieve fluorescence laparoscopy (FL): the excitation light source a Stryker L9000 LED lamp was filtered through a glass emission filter (Schott GG495) placed between 20(R)Ginsenoside Rg2 the laparoscope and the Stryker 1288 HD camera. Using the computer software system provided by Stryker (L9Calibration0.03DOT3) adjustments to the red blue and green components of the Stryker L9000 LED light source were made to allow visualization of the fluorescent tumors. A Stryker X8000 Xenon light source was used for bright field laparoscopy (BL) (Figure 1). Figure 1 Stryker laparoscopic setup. A standard laparoscopic tower was modified to achieve a fluorescence light mode that would permit detection of fluorescence signals while still allowing visualization of the background. The LED light source (Stryker L9000 LED … 20(R)Ginsenoside Rg2 Mouse laparoscopy Laparoscopy on mice was performed as previously described.18 19 A standard staging laparoscopy procedure was performed in which all four quadrants of the peritoneal cavity were examined in a systematic way. Each mouse was examined under both light modes fluorescence laparoscopy (FL) using the Stryker L9000 LED light source and bright light.